Pages

Friday, 30 April 2010

Individual's genome analyzed for risk of diseases, responses to treatmentFriday, 30 April 2010

For the first time, researchers have used a healthy person's complete genome sequence to predict his risk for dozens of diseases and how he will respond to several common medications. The risk analysis, from the Stanford University School of Medicine, also incorporates more-traditional information such as a patient's age and gender and other clinical measurements. The resulting, easy-to-use, cumulative risk report will likely catapult the use of such data out of the lab and into the waiting room of average physicians within the next decade, say the scientists.

"The $1,000 genome is coming fast," said cardiologist Euan Ashley, MD, assistant professor of medicine, referring to the cost of sequencing all of an individual's DNA.

"The challenge lies in knowing what to do with all that information. We've focused on establishing priorities that will be most helpful when a patient and a physician are sitting together looking at the computer screen."

Priorities will include whether a certain medication is likely to work for that particular patient, or if it is likely to have adverse side effects. Priorities that include ascertaining how a patient's obesity or smoking combine with his or her inherent genetic risk for - or protection against - heart attack or diabetes. In short, priorities that result in concrete clinical recommendations for patients based on a degree of data that has never existed before.

"We're at the dawn of a new age in genomics," said Stephen Quake, who is the Lee Otterson Professor of Bioengineering.

"Information like this will enable doctors to deliver personalized health care like never before. Patients at risk for certain diseases will be able to receive closer monitoring and more frequent testing, while those who are at lower risk will be spared unnecessary tests. This will have important economic benefits as well, because it improves the efficiency of medicine."

But it may also tell patients things they don't want to know.

Quake made national headlines last August when he used a technology he helped invent to sequence and publish his own genome for less than $50,000, and it is his genome that the researchers analyzed in this newest study. Ashley is the lead author of the research, which will be published in the May 1 issue of the Lancet.

An accompanying article about the ethical and practical challenges of such research, authored by a subset of the researchers involved in the first study, will appear in the online-only version of the Lancet on the same day. Hank Greely, JD, professor and director of Stanford's Center for Law and the Biosciences, is the senior author of the online piece.

"Patients, doctors and geneticists are about to hit by a tsunami of genome sequence data. The experience with Steve Quake's genome shows we need to start thinking - hard and soon - about how we can deal with that information," said Greely.

"When combined with other sources of information, genomics has the power to predict the diseases a person is most likely to develop and how he or she might respond to certain medicines," said Jeremy Berg, PhD, director of the NIH's National Institute of General Medical Sciences, which funded a portion of the works.

"This work provides a glimpse of how genomics can play a role in personalizing the medical care of individual patients."

The study began when the 40-year-old, seemingly healthy Quake asked Ashley's opinion about a particular snippet in his genome associated with an inherited disease called hypertrophic cardiomyopathy. People with the condition have enlarged hearts that do not beat effectively and are at risk for sudden cardiac death. Quake was interested because a distant relative had died unexpectedly in his sleep at the age of 19 - presumably from some type of heart problem. Ashley, who runs Stanford's Hypertrophic Cardiomyopathy Center, was alarmed.

"Given his family history and the particular genetic variation Steve has, I recommended that he be screened for the condition," said Ashley. Quake agreed, but the conversation got the two thinking about how to analyze the information in Quake's genome on a more global level.

"Several of us had already been thinking about how you would take someone's genomic profile, and translate what's in the billions of base pairs in that DNA to something that's clinically useful," said Ashley, who headed the group of geneticists, physicians, bioinformaticians and ethicists involved in the study.

"Then we realized, 'Hey, we already have someone's genome.'"

What's more, Atul Butte, MD, PhD, assistant professor in bioinformatics, and his lab members had already done a lot of the necessary leg work: They had spent the previous 18 months meticulously cataloguing publications that associated particular genetic changes called SNPs (for single nucleotide polymorphisms) with effects on specific diseases. It was the first time anyone had compiled all the information in one database.

"We read thousands of publications," said Butte, "and we made a list of every single spot in the genome where we know that, for example, the letter A raises the risk of a particular disease, or the letter T confers protection. And then came Steve with his genome, and we were ready."

Together the researchers designed an algorithm to overlay the genetic data upon what was already known about Quake's inherent risk - based on his age and gender - for 55 conditions, ranging from obesity and diabetes to schizophrenia and gum disease. For example, as a 40-year-old white male, Quake entered the study with a 16 percent chance of developing prostate cancer in his lifetime. But as the computer, based on Quake's genomic sequence, began to incorporate the data of study after study, his risk scorched first lower, and then higher. (The researchers weighted the contribution of each variant according to the number, and sample size, of published studies confirming the association.)

In the end, after incorporating information about 18 separate variants from 54 studies, they determined Quake's risk of prostate cancer is actually about 23 percent. The opposite is true for his risk of Alzheimer's disease, which began at 9 percent and ended - due to the presence of several protective variants - at about 1.4 percent. The scariest monsters in the closet, however, were obesity, type-2 diabetes and coronary artery disease, each of which Quake has a more than 50 percent chance of developing, and each of which can affect the development of the other.

Was it alarming? "It's certainly been interesting," said Quake of the findings.

"I was curious to see what would show up. But it's important to recognize that not everyone will want to know the intimate details of their genome, and it's entirely possible that this group will be the majority. There are many ethical, educational and policy questions that need to be addressed going forward."

Of course, a person's environment - in the form of choices he or she makes about diet, exercise and habits like smoking and drinking - can also powerfully affect disease risk. But if clinicians know that a patient has a higher-than-normal risk for a certain disease, they may recommend certain lifestyle changes more strongly.

"This opens the door to targeted environmental interventions based on a patient's genomics," said Butte.

"People who may want more control over their destiny could choose to exercise more, or eat better, or even avoid pesticides more conscientiously."

There's hope, too, in the promise of more effectively using available drugs to combat or prevent disease. Russ Altman, MD, PhD, is the principal investigator of the Stanford-managed Pharmacogenetics and Pharmacogenomics Knowledge Base, or PharmGKB - a curated, international data repository to help researchers understand how genetic variation among individuals contributes to differences in reactions to common medications. Quake's genome gave his group some new opportunities.

"With Steve, we thought, 'Let's apply everything we know about the effect of human genetic variation on drug response to his entire genome,'" said Altman, who together with Quake chairs Stanford's bioengineering department.

"And we came up with a table of drugs that are likely to work well for him, like statins, and others that he might need lower doses of, like warfarin."

The researchers also found five to 10 previously unknown SNPs in genes involved in drug response.

"This is really exciting because we never would have found these if we'd just relied on our usual panel of SNPs," said Altman.

"What's more, with whole-genome sequencing, you only ever have to do it once. Our understanding of the information will keep evolving, but the core data set doesn't change."

That evolving knowledge base will present a particular challenge, the researchers believe. Keeping people up-to-date on new findings involving genetic variants that they carry will be a tricky business. Clinicians of the future will walk a tightrope of informing people who have opted to have their genome sequenced of ongoing discoveries while also presenting the information as uncertain and likely to change. Furthermore, how shall we deal with the fact that a patient's genome by definition harbours information about that person's parents, children and other relatives who may not want to peek into their shared genomic crystal ball? Clearly, we have much with which to grapple.

"The world of medicine is going to change beyond belief," said Ashley.

"We are all going to have to learn how to deal with questions like these."

But what of Quake?
A complete physical pronounced him free of any sign of cardiomyopathy. But it also turned up somewhat elevated lipoprotein levels. Normally, given Quake's health and age, most physicians would take a watch-and-wait approach before recommending medication. However, in the face of this new information about Quake's lifetime genetic risk, and the likelihood, based on the pharmacogenetic data, that he would respond positively to statins, Ashley suggested he consider taking the cholesterol-lowering drugs. It is the first time anyone has ever made clinical recommendations based on a cumulative assessment of a patient's entire genome.

Jumping elements, some of which cause genetic diseases, become incorporated in the genome at different stages of human development Friday, 30 April 2010

The density of transposable (jumping) elements between sex chromosomes in primates may have important consequences for the studies of human genetic diseases, say Penn State University researchers. Erika Kvikstad, a 2009 Penn State Ph.D. graduate in genetics, and Kateryna Makova, an associate professor of biology at Penn State, used a statistical regression method to study the genomes of the human, chimpanzee, macaque, and orang-utans. They concluded that there is a strong sex-chromosome bias in the distribution of transposable elements, and providing insights about whether these non-coding, but important, DNA elements integrate themselves specifically into the male germline or female germline, or integrate themselves into the genome during the early stages of embryogenesis. Their study will be published in the May 2010 issue of the scientific journal Genome Research.

According to Kvikstad, now a postdoctoral scholar at the Université Claude Bernard Lyon 1 in Lyon, France, the team chose to study primates because of the importance of human evolution, human disease, and the "unique availability of a very detailed description of the human genome – more so than any other mammalian genome." The strides made in sequencing the human and other primate genomes have made this research possible only in the last decade. Makova, one of the researchers who contributed to the analysis of the macaque and chimpanzee genomes, notes that the sequence of the orang-utan genome used in the Penn State study has not yet been published. Makova received special permission to use the orang-utan data set in her study.

The team looked specifically at the densities of transposable elements, which are snippets of DNA capable of moving about, replicating themselves, and inserting copies within the genome. The classes of transposable elements are further distinguished by being short or long interspersed nuclear elements – SINEs and LINEs. Kvikstad and Makova looked at one SINE family – Alu sequences, which are about 300 base pairs long, – and one LINE family – L1 sequences, which can be thousands of bases long.

"The transposable elements that we chose to study, Alus and L1s, are significant because they are abundant," says Kvikstad.

"They comprise about a third of the primate genome. They are actively moving around in the genome via a copy-and-paste retro-transposition mechanism, so they can create new variation; for example, human diseases and cancers."

Even more importantly, Kvikstad points out:

"These transposable elements are highly abundant on the sex chromosomes – X and Y – which mean they could be evolving uniquely because of the unusual nature of sex-chromosome transmission. The Y is paternally inherited, so it resides in the male germline only; the X spends two-thirds of its time in the female germline and one-third in the male germline. If there are germline-specific differences in the activity of transposable elements, for example, we should see clues to these differences in their sex-chromosome distributions."

The team's findings surprised Kvikstad and Makova.

"Even after we corrected for regional genomic effects, we still observed a very strong sex-chromosome bias in distributions of transposable elements," says Makova.

"This finding clearly indicates that there are biases according to which elements integrate into the genome. There also are differences between these two classes of elements. Our study suggests that Alus probably integrate mostly in the male germline, while L1s integrate in both male and female germ lines, or they might integrate in early embryogenesis."

This bias has implications for understanding and perhaps someday even preventing and treating genetic diseases.

"For us to really understand how the genetic diseases occur, we need to know when the elements integrate – at what point in human development this occurs," says Makova.

"We are studying evolution mostly, but our results are relevant to genetic diseases caused by insertions of transposable elements in the genome. For instance, Alu insertions are known to cause some types of neurofibromatosis, haemophilia, breast cancer, Apert syndrome, cholinesterase deficiency, and complement deficiency."

When transposable elements were first discovered in the 1940s, many in the scientific community labelled them as "junk" DNA.

"I don't think many people agree that they are 'junk' DNA any longer. Many of these elements have function. Alu elements frequently possess the regulatory elements. Both the Alu and L1 elements are often involved in recombination, the phenomenon under which the genome can undergo rearrangement and reshuffling," says Makova.

Kvikstad and Makova spent a year analyzing the primate data. Previously, together with Francesca Chiaromonte, associate professor of statistics at Penn State, they had worked together on a project looking at primate insertions and deletions of a much smaller size, under 30 base pairs.

"We are the first team to look into this much detail at the distribution of transposable elements on human sex chromosomes," Makova says.

Kvikstad points out other important implications of this study.

"In particular, we noted that gene density was not a significant predictor of either Alu or L1 element density, at any evolutionary time point," she says.

"By contrast, density of conserved non-coding DNA or 'most conserved elements' was a strong negative predictor of L1 density – so L1 elements are scarce in regions of the genome that might contain many of these potentially functional non-coding DNAs. This is an important distinction, since previous studies inferring the action of natural selection in shaping the densities of transposable elements relied on gene density as a proxy for natural selection. Our results suggest that the potentially functional DNA residing in these most-conserved elements may be an additional hallmark of natural selection."

A comprehensive new gene expression study in embryonic stem cells has uncovered a transcription control mechanism that is not only more pervasive than once thought but is also heavily regulated by the cancer-causing gene c-Myc.

In research published in the April 30th edition of Cell, a team of Whitehead Institute for Biomedical Research researchers describes a pausing step in the transcription process that serves to regulate expression of as many as 80% of the genes in mammalian cells.

Scientists have long known that DNA-binding transcription factors recruit the RNA polymerase Pol II (which prompts copying of DNA into mRNA protein codes) to promoters in order to kick off the transcription process. Now researchers in the lab of Whitehead Member Richard Young have found that additional factors recruited to the promoters serve to stop transcription in its tracks shortly after it has begun.

"It's like the engine's running, but the transmission is not engaged on that transcription apparatus," says Young, who is also a professor of biology at MIT.

"You need something to engage that transmission."

It turns out that for a surprisingly large number of genes in embryonic stem cells, that "something" is the transcription factor c-Myc. This so-called pause release role for c-Myc is significant, as many of c-Myc's targets are genes in highly proliferative cells. Over-expression of c-Myc is a hallmark of a number of tumours, and it now appears that c-Myc's ability to release transcriptional pausing is linked with the hyper-proliferation that is characteristic of cancer cells.

"Our findings provide the molecular basis for loss of proliferation control in some cancers," says Peter Rahl, a postdoctoral researcher in Young's lab and first author of the Cell paper.

Armed with this new understanding of c-Myc's role in controlling proliferation genes, Young and his colleagues have embarked on a search for drugs that could interrupt c-Myc's pause-release activity in tumours where it is over-expressed.

"Clearly, cancer cells are able to exploit mechanisms that normally operate in embryonic stem cells, so I expect further understanding of embryonic stem cell control mechanisms will give us additional insights into human disease mechanisms." says Young.

A team of scientists led by the Department of Energy's Joint Genome Institute (JGI) and the University of California, Berkeley, is publishing this week the first genome sequence of an amphibian, the African clawed frog Xenopus tropicalis, filling in a major gap among the vertebrates sequenced to date.

"A lot of furry animals have been sequenced, but far fewer other vertebrates," said co-author Richard Harland, UC Berkeley professor of molecular and cell biology.

"Having a complete catalogue of the genes in Xenopus, along with those of humans, rats, mice and chickens, will help us reassemble the full complement of ancestral vertebrate genes."

The high-quality draft sequence of the genome of X. tropicalis, often called the Western clawed frog, will also aid researchers who now use the frog's more popular cousin, Xenopus laevis (zen'-uh-pus lay'-uh-vus), to study embryo development and cell biology. X. laevis, with its large and easily manipulated eggs, has told scientists a lot about how a fertilized egg develops into an embryo, including how embryos set up front-back and head-tail axes. The genome of tropicalis will help scientists connect genetic changes with developmental milestones in both species, Harland said.

"Xenopus has been among the last model organisms to be sequenced," after the mouse, chicken, nematode, zebrafish and fruit fly, he said.

"It will be tremendous to have a high quality sequence of X. tropicalis upon which to build the X. laevis sequence."

"The availability of the Xenopus genome also opens up the possibility of studying the effect of endocrine disruptors at the molecular and genomic level," added first author Uffe Hellsten, a bioinformaticist at the JGI. These chemicals mimic frogs' own hormones, and their presence in lakes and streams may be partly responsible for the decline of frog populations worldwide.

"Hopefully, understanding the effects of these hormone disruptors will help us preserve frog diversity and, since these chemicals also affect humans, could have a positive effect on human health," he added.

Hellsten, Harland and 46 other scientists from 24 institutions will publish the draft genome sequence and genome-wide analysis in the April 30 issue of the journal Science.

Xenopus, meaning "strange foot," is a genus of more than 20 frog species native to sub-Saharan Africa. When biologists discovered in the early 20th century that these frogs were unusually sensitive to human chorionic gonadotropin (HCG), they were adopted widely as a low-cost pregnancy test in hospitals, primarily in the 1940s and '50s. Inject a frog with a woman's urine and, it she is pregnant, the HCG in the urine will make the frog ovulate and produce eggs in 8-10 hours.

Imported from South Africa, these frogs were kept in hospitals around the world, where scientists soon discovered their value in studying embryo development. Their large eggs also were easy to inject with chemicals; making them big spherical test tubes, Harland said. Plus, the frogs could be induced to lay eggs at any time of year by injecting them with hormones.

When the Joint Genome Institute decided to sequence a frog genome, however, the Xenopus research community recommended X. tropicalis over X. laevis because tropicalis has half the genome size. While X. tropicalis is diploid, with two copies of each gene on 10 pairs of chromosomes, the X. laevis genome has undergone duplication and could have four copies of every gene on 18 pairs of chromosomes. Sequencing X. laevis would have been not only more costly, but also harder, because of the difficulty of matching genes to the proper chromosome.

Nevertheless, the high quality draft sequence will provide a "scaffold upon which to assemble the X. laevis genome," Harland said. Harland and Daniel Rokhsar, co-lead of the Xenopus genome project, UC Berkeley biology professor and JGI scientist, have applied for a National Institutes of Health grant to sequence the X. laevis genome. Harland said that, because of faster and cheaper sequencing machines, the cost would be about $1 million – about 20 times less than the cost of sequencing the X. tropicalis genome over a three-year period starting in 2002.

Though the draft genome sequence has been available to scientists for several years, the new paper is the first analysis of the full genome. According to Hellsten, a comparison of regions around specific genes in the frog, chicken and human genomes shows that they are amazingly similar, indicating a high level of conservation of organization, or structure, on the chromosomes.

"When you look at segments of the Xenopus genome, you literally are looking at structures that are 360 million years old and were part of the genome of the last common ancestor of all birds, frogs, dinosaurs and mammals that ever roamed the earth," said Hellsten.

"Chromosome archaeology helps to understand the history of evolution, showing us how the genetic material has rearranged itself to create the present day mammalian genome and present day amphibian genome."

"The rat and mouse genomes gave us the impression that genomes evolve quickly, but that turns out to be characteristic of rodents, not of all organisms," Harland said.

"Instead, it seems that breaking and rearranging chromosomes are extremely rare events in evolution."

The frog genome also contains genes similar to at least 1,700 genes that, in humans, are associated with disease. Thus, understanding these genes in frogs could help biologists understand how they are involved in human disease, Hellsten said.

The X. tropicalis genome, which contains more than 20,000 genes – humans have about 23,000 – is of particular interest to Harland, who is part of a small community of scientists studying this frog in addition to its larger cousin, X. laevis. The frogs take up less room and have a shorter lifecycle – as little as 4 months instead of a year or more – while their smaller eggs are still relatively easy to manipulate and inject. The sequence will speed up the adoption of X. tropicalis for genetic studies in addition to developmental and cell biological studies, he said.

Harland injects eggs with nucleic acids that activate or block the action of specific genes or their protein products in order to discover their function.

"If you want to knock down multiple gene products, it's a much simpler exercise to knock them down in tropicalis, with only two copies of each gene, as opposed to knocking down or targeting gene products in laevis, where the problem is twice as complex," he said.

"Now that we have a complete catalogue of genes, we can also design a gene chip to look at the changes in gene expression across the whole genome, whereas previously we were restricted to the examples people had chosen to study."

Thursday, 29 April 2010

First step in effort to use bone stem cells to repair malformed, damaged bone Thursday, 29 April 2010

Working with mice, a team of researchers has pinpointed the location of bone generating stem cells in the spine, at the ends of shins, and in other bones. The team also has identified factors that control the stem cells' growth. The research was conducted at the National Institutes of Health and other institutions.

"Identifying the location of bone stem cells and some of the genetic triggers that control their growth is an important step forward," said Alan E. Guttmacher, M.D., acting director of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the NIH institute where much of the research took place.

"Now, researchers can explore ways to harness these cells so that ultimately they might be used to repair damaged or malformed bone. Also, studies of this stem cell population could yield insight into the formation of bone tumours."

Researchers have long known that stem cells from bone marrow give rise to bone cells and to red and white blood cells. The current study is the first to identify the location of bone stem cells in the adult mouse skeleton. The researchers refer to the newly identified cells as bone stromal cells. "Stroma" is a term used to describe a supportive or connective structure in biological tissue. The term distinguishes the cells from hematopoietic stem cells, which give rise to blood cells, and which are found in bone marrow.

The findings appear online in the Proceedings of the National Academy of Sciences. The study's first author was NICHD predoctoral fellow Kit Man Tsang, a graduate student of the Chinese University of Hong Kong. Tsang's thesis advisor and the senior author of the study was Constantine A. Stratakis, M.D., D.Sc., acting director of the NICHD Division of Intramural Research. Other authors of the study were from the NICHD; the NIH's Division of Veterinary Resources and the National Institute of Dental and Craniofacial Research; as well as the Johns Hopkins University School of Medicine and The Ohio State University.

The researchers undertook the study to learn more about the role of two genes, dubbed Prkar1a and prkaca, in a key chemical sequence that provides energy to cells. Prkar1a has been implicated in a variety of rare human cancers, of the bone, nervous system, and thyroid. When the two genes are working normally, bone cell growth proceeds normally and cancerous overgrowth is kept in check. In previous research, the researchers learned that tumours formed in numerous tissues when they inactivated prkar1a.

In the current study, they inactivated one copy each of the two genes. Like human beings, mice have two copies of most genes. The mice in the study had one functioning copy each of prkar1a and prkaca and one non-functioning copy of each gene.

The researchers predicted that disabling only one copy of each gene would offer protection from bone tumour growth. In fact, the combination had the opposite effect. Tumourous growths were unexpectedly prolific and developed much earlier than expected: Tumours appeared in mice as young as 3 months old, compared with 6 to 9 months old in the previous studies, in which only prkar1a had been inactivated. Moreover, abnormal growths formed near cartilage in the legs, along the tail, and the remaining vertebrae of the mice.

The bone tumours were also more extensive in the mice with the two inactivated genes compared with their counterparts having only a single inactivated gene. All mice with the two mutations had abnormal growths on their tail bones by the time they were 9 months old and showed abnormalities in their vertebrae by 12 months.

Examination of the tumour cells and of cells from the same locations in mice that did not have tumours confirmed that the cells were bone stem (stromal) cells. Specifically, proteins on the surface of the cells were identical to proteins found on other types of stem cells. Moreover, the tumours formed only at locations where bone is actively growing, even in the adult mouse skeleton.

"We didn't notice abnormal growth in the skull, for example," said Dr. Stratakis.

The findings open up two avenues for additional research. Studies to identify the chemical signals that initiate the formation of new bone tissue could lead to new techniques for regenerating damaged or injured bone. Similarly, studies of the chemical events that trigger the initial stages of tumour formation may lead to ways to prevent or treat bone tumours.

General information about stem cells is available on the NIH Web site.
.........

Scientists have for the first time succeeded in extracting vital stem cells from sections of vein removed for heart bypass surgery. Researchers funded by the British Heart Foundation (BHF) found that these stem cells can stimulate new blood vessels to grow, which could potentially help repair damaged heart muscle after a heart attack.

The research, by Paolo Madeddu, Professor of Experimental Cardiovascular Medicine and his team in the Bristol Heart Institute (BHI) at the University of Bristol, is published in the leading journal Circulation.

Around 20,000 people each year undergo heart bypass surgery. The procedure involves taking a piece of vein from the person's leg and grafting it onto a diseased coronary artery to divert blood around a blockage or narrowing.

The surgeon normally takes out a longer section of vein than is needed for the bypass. The Bristol team successfully isolated stem cells from leftover veins that patients had agreed to donate.

In tests in mice, the cells proved able to stimulate new blood vessels to grow into injured leg muscles. Professor Madeddu and his team are now beginning to investigate whether the cells can help the heart to recover from a heart attack.

"This is the first time that anyone has been able to extract stem cells from sections of vein left over from heart bypass operations," Professor Madeddu said.

"These cells might make it possible for a person having a bypass to also receive a heart treatment using their body's own stem cells.”

"We can also multiply these cells in the lab to make millions more stem cells, which could potentially be stored in a bank and used to treat thousands of patients."

Professor Peter Weissberg, Medical Director of the BHF, said:

"Repairing a damaged heart is the holy grail for heart patients. The discovery that cells taken from patients' own blood vessels may be able to stimulate new blood vessels to grow in damaged tissues is a very encouraging and important advance. It brings the possibility of 'cell therapy' for damaged hearts one step closer and, importantly, if the chemical messages produced by the cells can be identified, it is possible that drugs could be developed to achieve the same end."
.........

Sunday, 25 April 2010

Scientists may be one step closer to being able to generate any type of cells and tissues from a patient's own cells. In a study that will appear in the journal Nature and is receiving early online release, investigators from the Massachusetts General Hospital Center for Regenerative Medicine (MGH-CRM) and the Harvard Stem Cell Institute (HSCI), describe finding that an important cluster of genes is inactivated in induced pluripotent stem cells (iPSCs) that do not have the full development potential of embryonic stem cells. Generated from adult cells, iPSCs have many characteristics of embryonic stem cells but also have had significant limitations.

"We found that a segment of chromosome 12 containing genes important for fetal development was abnormally shut off in most iPSCs," says Konrad Hochedlinger, PhD, of the MGH-CRM and HSCI, who led the study. Hochedlinger is an associate professor in the Harvard University Department of Stem Cell and Regenerative Medicine.

"These findings indicate we need to keep improving the way we produce iPSCs and suggest the need for new reprogramming strategies."

Although iPSCs appear quite similar to embryonic stem cells and give rise to many different types of cells, they have important limitations. Several molecular differences have been observed, particularly in the epigenetic processes that control which genes are expressed, and procedures that are able to generate live animals from the embryonic stem cells of mice are much less successful with iPSCs.

Previous studies have compared iPSCs generated with the help of viruses, which can alter cellular DNA, to embryonic stem cells from unrelated animals. To reduce the chance that the different sources of the cells were responsible for observed molecular differences, the MGH/HSCI research team prepared two genetically matched cell lines. After generating mice from embryonic stem cells, they used a technique that does not use viruses to prepare lines of iPSCs from several types of cells taken from those animals. They then compared the iPSCs with the original, genetically identical embryonic stem cells.

The most stringent assay of cells' developmental potential showed that two tested lines of embryonic stem cells were able to generate live mice as successfully as in previous studies, but no animals could be generated from genetically matched iPSCs. Closely comparing RNA transcription profiles of several matched cell lines revealed significantly reduced transcription of two genes in the iPSCs. Both genes are part of a gene cluster on chromosome 12 that normally is maternally imprinted – meaning that only the gene copies inherited from the mother are expressed.

Examination of more than 60 iPSCs lines developed from several types of cells revealed that this gene cluster was silenced in the vast majority of cell lines. While the gene-silenced iPSCs were able to generate many types of mouse tissues, their overall developmental potential was limited. In an assay that produces chimeric animals that incorporate cells from two different stem cells, mice produced from gene-silenced cells had very few tissues that originated from the iPSCs. However, in a few iPSC lines, the gene cluster was normally activated, and in the most rigorous developmental assay, those iPSCs were as successful in producing live animals as embryonic stem cells have been. The authors believe this is the first report of animals being produced entirely from adult-derived iPSCs.

"The activation status of this imprinted cluster allowed us to prospectively identify iPSCs that have the full developmental potential of embryonic stem cells," says Matthias Stadtfeld, PhD, of the MGH-CRM and HSCI, a co-lead author of the report.

"Identifying pluripotent cells of the highest quality is crucial to the development of therapeutic applications, so we can ensure that any transplanted cells function as well as normal cells. It's going to be important to see whether iPSCs derived from human patients have similar differences in gene expression and if they can be as good as embryonic stem cells – which continue to be the gold standard – in giving rise to the 220 functional cell types in the human body."

Hochedlinger adds:

"Previous studies in mice have shown that embryonic stem cells derived from nuclear transfer – the technique used to clone animals – are indistinguishable from stem cells derived from fertilized embryos. Nuclear transfer is another way of reprogramming adult cells into embryonic-like cells, and comparing that approach with iPSC generation may yield important insights into ways of producing the safest and highest quality pluripotent cells for use in patients."

Wednesday, 21 April 2010

Singapore scientists make breakthrough findings on early embryonic developmentWednesday, 21 April 2010

Scientists at the Genome Institute of Singapore (GIS) have recently generated significant single cell expression data crucial for a detailed molecular understanding of mammalian development from fertilization to embryo implantation, a process known as the preimplantation period. The knowledge gained has a direct impact on clinical applications in the areas of regenerative medicine and assisted reproduction.

This study, published in Developmental Cell on April 20, 2010, is the first of its kind to apply single cell gene expression analysis of many genes to hundreds of cells in a developmental system.

Using the new BioMark microfluidic technology and the mouse preimplantation embryo as a model, the scientists were able to study the expression of 48 genes from individual cells and applied this to analyze over 600 individual cells from the 1-cell to the 64-cell stage of preimplantation development.

This high throughput single cell research methodology provides the scientists with the ability to detect dynamic patterns in cellular behaviour, which is unprecedented in the field. Significantly, the findings of the study resolves some of the arguments pertaining to cellular differentiation events and places fibroblast growth factor signalling as the primary event in the later cell fate decisions.

Executive Director at the GIS, a biomedical research institute of the Agency for Science, Technology and Research (A*STAR), Professor Edison Liu said:

"This remarkable work by Guoji Guo, Mikael Huss, Paul Robson and colleagues uses new microgenomic technologies to map, over time, how a single cell decides to permanently become different parts of an embryo. Within one division, cells commit to specific developmental lineages by expressing defined sets of genes. This research now opens the possibility of assessing the genetic triggers for fate determination of individual cells in developmental time. On another level, this work highlights the importance of new micro-technologies in advancing the understanding of early embryonic events."

"This is a real technological tour de force. The authors investigated changes in expression of multiple genes on the single cell level during preimplantation mouse development. They clearly demonstrated gradual and stochastic lineage allocation and absence of predetermination. These results conclusively resolved one of the hotly debated issues in mammalian development and provided important new insight into the mechanism which regulates early development in mammals."

"These are important findings. The team at GIS provided a new look into the complex and little-understood process of early embryo development. It also demonstrates the power of single cell gene expression. It is clear that individual cells and small groups of cells behave differently than the aggregate population, and these differences are key to understanding the biology of the system as a whole." said Gajus Worthington, president and chief executive officer of Fluidigm.

"It always provides a special thrill when researchers use the capabilities of Fluidigm's technology to bring insight to the body of scientific knowledge."

The preimplantation period involves the first cellular differentiation events in mammalian development including the formation of pluripotent cells from where embryonic stem (ES) cells are derived. Being one of the simplest mammalian developmental systems to study, it can provide comprehensive understanding of the complex molecular control of reprogramming and cell fate decisions.

Tuesday, 20 April 2010

Findings suggest that new genome assemblies based solely on next-generation sequencing might miss many of these sitesTuesday, 20 April 2010

Researchers have discovered 2,363 new DNA sequences corresponding to 730 regions on the human genome by using new approaches. These sequences represent segments of the genome that were not charted in the reference map of the human genome.

"A large portion of those sequences are either missing, fragmented or misaligned when compared to results from next-generation sequencing genome assemblies on the same samples," said Dr. Evan Eichler, senior author on the findings published April 19 in the advanced online edition of Nature Methods. Eichler is a University of Washington (UW) professor of genome sciences and an investigator with the Howard Hughes Medical Institute.

"These findings suggest that new genome assemblies based solely on next-generation sequencing might miss many of these sites."

Dr. Jeffrey M. Kidd was lead author of the article, which described the new techniques the research team used to find some of the missing sequences.

Kidd headed the study while earning his Ph.D. at the University of Washington in the Eichler lab. Kidd is now a postdoctoral fellow at Stanford University.

"Over the past several years, the extent to which the structure of the genome varies among humans has become clearer. This variation suggested that there must be portions of the human genome where DNA sequences had yet to be discovered, annotated and characterized," he said

"We hope that these sequences ultimately will be included as part of future releases of the reference human genome sequence."

The reference genome is a yardstick – or standard for comparison – for studies of human genetics.

The human reference genome was first created in 2001 and is updated every couple of years, Kidd explained. It is a mosaic of DNA sequences derived from several individuals. He went on to say that about 80 percent of the reference genome came from eight people. One of them actually accounts for more than 66 percent of the total.

Along with their collaborators at Agilent, the team designed ways to examine these newly identified sequences in a panel of people representing populations from around the world. The researchers found that, in some cases, the number of copies of these sequences varied from person to person.

The fact that a person can have one or more copies, or no copy at all, of a particular DNA sequence may account for why these sequences were missing from the reference genome. The researchers also found that some of these sequences were common or rare in different populations, depending on from which part of the globe their ancestors originated.

"Each segment of the reference genome is from a single person, and reflects the genome of that individual. If the donor sample was missing a sequence that many other people have, that sequence would not be represented in the reference genome." Kidd explained.

"That is why some of the positions on the reference genome represent rare structural configurations or entirely omit sequences found in the majority of people."

Kidd said that the study published in Nature Methods used information from nine individuals, representing various world populations, to search for and fill in some of the missing pieces.

By looking at genomes from seven kinds of animals, the researchers were also able to show that some of the newly identified DNA sequences appear to have been conserved during the evolution of mammals and man. The animals whose genomes were studied were chimpanzee, Bornean orang-utan, Rhesus monkey, house mouse, Norway rat, dog, and horse.

"Some of the sequences were present in several different species, but were absent from the reference genome," Kidd said.

"Some of the sequences present in several mammals actually correspond to sites of variations in humans – some people have retained a particular sequence, and others have lost it."

The researchers also developed a method to accurately genotype many of the newly found DNA sequences and created a way to look at variations in the number of copies of these sequences, thereby opening up regions of the human genome previously inaccessible to such studies.

"Scientists can now begin trying to understand the functional importance of these sequences and their variations," Kidd said.

The 1,000 Genomes Project (an international effort to fully sequence the genomes of a thousand anonymous individuals) and other genome studies are amassing massive amounts of data on DNA sequences that are then mapped to the reference genome, he added. Any study, he continued, that improves the completeness and quality of the reference genome assembly will thereby benefit these projects and lead to a fuller picture of the extent of human genomic variation.

Biomechanical properties similar to normal tissueTuesday, 20 April 2010

Scientists from the University of Granada, Spain, have generated artificial human skin by tissue engineering based on agarose-fibrin biomaterial. The artificial skin was grafted onto mice, and optimal development, maturation and functionality were obtained. This pioneering finding will allow the clinical use of human skin and its use in many laboratory tests on biological tissues – which, additionally, would avoid the use of laboratory animals. Further, this finding could be useful in developing new treatment approaches for dermatological pathologies.

This research was conducted by José María Jiménez Rodríguez, from the Tissue Engineering Research group of the Department of Histology of the University of Granada, and coordinated by professors Miguel Alaminos Mingorance, Antonio Campos Muñoz and José Miguel Labrador Molina.

Researchers from the University of Granada firstly selected the cells that would be employed in generating artificial skin. Then, they analysed the evolution of the in-vitro culture and, finally, they performed a quality control of the tissues grafted onto nude mice. To this purpose, several immunofluorescence microscopy techniques had to be developed. These techniques allowed researchers to evaluate such factors as cell proliferation, the presence of differentiating morphological markers, the expression of cytokeratin, involucrine and filaggrin, angiogenesis and artificial skin development into the recipient organism.

Human Skin Samples
To make this assay, researchers obtained human skin from small biopsies belonging to patients following surgery at the Plastic Surgery Service of the University Hospital Virgen de las Nieves in Granada. All patients gave their consent to take part in this research study.

To create artificial human skin, human fibrin from plasma of healthy donors was used. Researchers then added tranexamic acid, to prevent fibrinolysis, and calcium chloride to precipitate fibrin coagulation, and 0.1% agarose. These artificial-skin substitutes were grafted on the back of the nude mice, with the purpose of observing its evolution in vivo. The equivalent skin substitutes were analysed by transmission and scanning light and electron microscopy and immunofluorescence.

The skin created in the laboratory showed adequate biocompatibility rates with the recipient and no rejection, separation of wound edges or infection was registered. Additionally, the skin of all animals used in the study started to show granulation after six days from implantation. Within the following twenty days, scarring was complete.

The experiment conducted by the University of Granada is the first to create artificial human skin with a dermis made of fibrin-agarose biomaterial. To this date, artificial skin substitutes were elaborated with other biomaterials as collagen, fibrin, polyglycolic acid, chitosan, etc..

These biomaterials "added resistance, firmness and elasticity to the skin" – according to Prof. Jiménez Rodríguez.

"Definitively, we have created a more stable skin with similar functionality to normal human skin."
.........

Monday, 19 April 2010

Cell Transplants May Benefit Children with Cerebral PalsyMonday, 19 April 2010

A unique cell type that supports and surrounds (ensheathes) neurons within the nose (olfactory system) known as olfactory ensheathing cells (OECs), possess the ability to regenerate, are relatively easy to obtain, and have become prime candidates for transplantation to repair a number of lesions in the central nervous system (CNS). Transplanted OECs, known to retain exceptional plasticity and promote olfactory blood vessel growth while offering neuroprotection, have been demonstrated to be potentially useful for a number of neurological disorders, including multiple sclerosis, spinal cord injury and amyotrophic lateral sclerosis (ALS).

A group of Chinese researchers hypothesized that OECs might also hold promise for treating cerebral palsy (CP), a neurological disorder appearing in infancy or early childhood and characterized by its permanent effects on muscle movement. The study is published in issue 19(2) of Cell Transplantation.

"CP is a class of brain lesion in children with a wide variety of causes - from abnormal brain development to peri-natal injuries - and manifesting in progressive physical dysfunction," said corresponding author Dr. Hongyun Huang of the Beijing Rehabilitation Center.

"We conducted a randomized, controlled clinical trial with 33 volunteers, 14 of whom completed the six-month study, to determine if transplanted OECs were effective in treating children and adolescents with CP, given that CP shares many of the same features of other degenerative diseases."

According to the researchers, 83 percent of the children with CP that they examined had abnormal radiological findings, with white matter damage being the most common abnormality. Tissue loss, inadequate or delayed myelination, glial scars and shrunken white matter of the brain were also encountered. The white matter is made up of nerve fibres communicating between brain areas.

The research team's hypothesis and protocol was developed with prior knowledge of a key location in the brain's frontal lobes (defined as the "Key Point for Neural network Restoration (KPNNR)" based on previous studies) for injecting OECs and that the injected OECs would produce Schwann cell-like myelin sheaths around demyelinated axons.

Results were measured by both the Gross Motor Function Measure (GMFM-66) and the Caregiver Questionnaire Scale.

"This trial, albeit small in sample size, indicates that OEC KPNNR transplantation may be effective for functional improvement in children and adolescents with CP," said Dr. Huang.

"Our results showed that transplanting OECs into CP patients could improve the neurological function of the patients and did not cause significant side effects. The procedure may be a plausible method to treat this as yet incurable disorder."

"In parallel with recently FDA-approved US clinical trials of cell therapy for adult stroke and cerebral palsy, this clinical study in China advances the use of stem cells for treating brain disorders, but a very careful assessment of this experimental treatment needs to be exercised in order to gauge its safety and efficacy," says Cell Transplantation associate editor Dr. Cesar V. Borlongan.

Friday, 16 April 2010

In a multidisciplinary approach, Professor Yves Barral, from the Biology Department at ETH Zurich and the computer scientists Dr. Gina Cannarozzi and Professor Gaston Gonnet, from the Computer Science Department of ETH Zurich and the SIB Swiss Institute of Bioinoformatics, joined forces to chase possible sub-codes in genomic information. The study, which will be published in today's issue of the journal Cell, led to the identification of novel sequence biases and their role in the control of genomic expression.

Each cell of an organism contains a copy of its genome, which is a sequence of deoxyribonucleotides, also called DNA. The cell is able to translate some of the coding sequences into different proteins, which are necessary for an organism's growth, the repair of some tissues and the provision of energy. For this translation work, the cell follows a decoding procedure provided by the "genetic code", which tells what protein is made from a given sequence. The genetic code has been known since the early 1960's.

The researchers from ETH and SIB now identified a new sub-code that determines at which rate given products must be made by the cell. This information has several interesting implications. First, it provides novel insights into how the decoding machinery works. Secondly, and more pragmatically, it makes possible to read information about gene expression rates directly from genomic sequences, whereas up to now, this information could only be obtained through laborious and expensive experimental approaches, such as microarrays.

"A cell must respond very quickly to injuries such as DNA damage and to potent poisons such as arsenic. The new sub-code enables us to know which genes are turned-on quickly after these insults and which are best expressed slowly. One benefit of this study is that we now can get this information using only analysis of the coding sequence", said Dr. Gina Cannarozzi.

Additionally, the new sub-code provides insight into cellular processes at the molecular level. In every living cell, the translation allowing the production of proteins takes place at specialised factories, the ribosomes. The discovery of this novel sub-code will therefore also provide more information about the functioning of these ribosomes. Indeed, all the data gathered up to now indicate that these factories recycle their own components, the tRNAs, to optimize the speed of protein synthesis. This discovery of a new way to regulate translation could potentially be exploited to more efficiently produce therapeutic agents and research reagents. For example, many therapeutic agents, such as insulin, are produced by expressing a protein in a foreign host such as E. coli or S. cerevisiae. The new sub-code can be now used to rewrite the information such as to optimize in a much more rational manner the amount of product delivered by the foreign host.

Finding is expected to steer future work on therapies down the most efficient and promising paths Friday, 16 April 2010

Researchers in China are reporting that they have found a way to determine which somatic cells – or differentiated body cells – that have been reprogrammed into a primordial, embryonic-like state are the most viable for therapeutic applications.

In a paper published online last week by the Journal of Biological Chemistry, two collaborating teams from institutes at the Chinese Academy of Sciences point to a marker they found in induced-pluripotent stem cells, or iPS cells, taken from mice. That marker is a cluster of small RNA whose expression appears strictly correlated with levels of pluripotency, or "stemness." (The more pluripotent, the more likely a stem cell will develop into the desired tissue, organ or being.)

"We identified a genomic region encoding several genes and a large cluster of microRNAs in the mouse genome whose expression is high in fully pluripotent embryonic stem cells and iPS cells but significantly reduced in partially pluripotent iPS cells, indicating that the Dlk1-Dio3 region may serve as a marker," said Qi Zhou, a researcher at the CAS Institute of Zoology and co-author of the paper.

"No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotent levels."

After the creation of the first iPS cells in Japan in 2006, Zhou and others set out to determine whether the reprogrammed adult cells are versatile enough to generate an entire mammalian body, as embryonic stem cells can.

Then, last summer, Zhou announced that his team had reprogrammed somatic cells of mice, injected them into embryos and created 27 live offspring, which clearly demonstrated that iPS cells can, like embryonic stem cells, produce healthy adults. Though lauded as a huge step forward, they also found not all iPS cells were perfect: Many of the iPS cell lines used did not produce mice, and some of the mice that were produced had abnormalities.

"The success rate of obtaining iPS cells with full pluripotency was still extremely low, which significantly hindered the application of iPS cells in therapeutics and other aspects," Zhou said.

Believing that there might be some intrinsic gene expression difference between the lines of iPS cells with varying levels of pluripotency that could be identified at early culture stages, so that less viable lines could be abandoned and more viable lines focused on, Zhou teamed up with bioinformatics specialist Xiu-Jie Wang, who works at the Chinese academy's Institute of Genetics and Developmental Biology.

Together, their groups profiled the small RNA expression patterns of ES and iPS cell lines from different genetic backgrounds and with different pluripotent levels using Solexa technology.

"There are nearly 50 miRNAs encoded in this region, and those expressed miRNAs all exhibited consistent and significant expression differences between stem-cell lines with different pluripotency levels," Wang said.

"With this discovery, iPS cells with different pluripotency can be distinguished in their early phases, which will, thus, significantly improve the production of full pluripotent iPS cells and promote their application in disease therapy," Wang said.

As stem cells can be applied in the treatment of many diseases related to tissue replacement or organ implantation, Zhou said, if the team's findings also are true for humans, "it will cause a revolution in stem-cell research and the application of it in the very near future."

Thursday, 15 April 2010

A new technique for reprogramming human adult cells could greatly improve the safety and efficiency of producing patient-specific stem cells for use in a range of therapeutic applications to repair or replace damaged or diseased tissues. A description of this innovative strategy is published in the peer-reviewed journal Cellular Reprogramming, published by Mary Ann Liebert, Inc. The paper is available free online.

Stem cells offer great promise for use in cellular therapy to regenerate specific cell populations in the body. The ability to derive stem cells from a patient's own tissue eliminates the risk that they will stimulate an immune response and be rejected after transplantation. The process for transforming adult somatic cells into a form of stem cells called induced pluripotent stem (iPS) cells, which are capable of differentiating into all three major cell types, involves reprogramming the cell's genetic contents. This is usually achieved using a virus to introduce multiple (typically four) genetic factors, called transcription factors, into the cells; these would include an oncogene, which carries the risk of cancerous transformation.

This new method was applied successfully to reprogram cells from the human gut to form iPS cells, as described in the article "Generation of Human-Induced Pluripotent Stem Cells from Gut Mesentery-Derived Cells by Ectopic Expression of OCT4/SOX2/NANOG." As the title states, this novel technique required only three transcription factors and no oncogenes. The authors, Yang Li, Hongxi Zhao, Feng Lan, Andrew Lee, Liu Chen, Changsheng Lin, Yuanqing Yao, and Lingsong Li, from Peking University and the Fourth Military Medical University, Tangdu Hospital, in the People's Republic of China, and Stanford University School of Medicine, in Stanford, California, demonstrated the pluripotency of these gut-derived stem cells and their ability to form cell types of all three germ layers.

"These observations are very exciting because they demonstrate that a different cell type can be used when making iPS cells.”

In addition, Lingsong Li and his colleagues show that Nanog has a key role when reprogramming these cells, whereas it does not with other cell types.

“This difference provides us with an important new opportunity to study the mechanisms involved in reprogramming," says Professor Sir Ian Wilmut, OBE, FRS, FRSE, Editor-in-Chief of Cellular Reprogramming and director of the MRC Centre for Regenerative Medicine in Edinburgh.
.........

Tuesday, 13 April 2010

The groundbreaking new findings will speed research on potential therapiesTuesday, 13 April 2010

Scientists at The Scripps Research Institute have solved the decade-old mystery of why human embryonic stem cells are so difficult to culture in the laboratory, providing scientists with useful new techniques and moving the field closer to the day when stem cells can be used for therapeutic purposes.

The research is being published in the journal Proceedings of the National Academy of Sciences (PNAS) during the week of April 12, 2010.

"This paper addresses a long-standing mystery," said Scripps Research Associate Professor Sheng Ding, who is senior author of the paper.

"Scientists have been puzzled by why human embryonic stem cells die at a critical step in the culture process. In addition to posing a question in fundamental biology, this created a huge technical challenge in the lab."

The new paper, however, provides elegant solutions to both aspects of this problem.

In the study, the team discovered two novel synthetic small molecule drugs that can be added to human stem cell culture that each individually prevent the death of these cells. The team also unravels the mechanisms by which the compounds promote stem cell survival, shedding light on a previously unknown aspect of stem cell biology.

Notorious Fragility
The hope of most researchers in the field is that one day it will be possible to use stem cells — which possess the ability to develop into many other distinct cell types, such as nerve, heart, or lung cells — to repair damaged tissue from any number of diseases, from Type 1 diabetes to Parkinson's disease, as well as from injuries.

Laboratory work with human embryonic stem cells, however, has been hampered by their notorious fragility. In the process of growing stem cells in culture, scientists must split off cells from their cell colonies. At this point in the process, however, human embryonic stem cells die unless the scientists take extraordinary care that this does not happen.

"The current techniques to keep these cells alive are tedious and labour-intensive," said Ding.

"Keeping the cells alive is so difficult that some people are discouraged from entering the field. It is very frustrating experience for everyone."

Mysteriously, mouse embryonic stem cells — which share much basic biology with human embryonic stem cells — do not pose the same difficulties in the laboratory. They can usually be split off from a colony and go on to survive and thrive.

To address these issues, the scientists decided to start with a screen of a library of chemical compounds to see if they could find any small molecules that could be added to the human embryonic stem cell culture that would promote the cells' survival.

When the scientists examined their results, they were elated to find two novel compounds (named Thiazovivin and Pyrintegrin) that both worked to dramatically protect the cells, promoting human embryonic stem cell survival by more than 30 fold.

"Basically, this solved this cell survival problem that has been plaguing scientists for more than 10 years," said Ding.

The Importance of Interaction
But the scientists did not stop there.

Next, using the two new survival-promoting small molecules as clues, the scientists set out to understand the biological mechanism behind the cells' survival or demise. By examining cell growth in the presence and absence of the compounds, the team found that the key factor was a protein on the cell surface called e-cadherin, which mediates interactions among cells and between cells and the extracellular matrix (a structure present between a variety of animal cells that provides support and anchorage for cells and regulates intercellular communication).

"While in the past people have often talked about the proteins in cell nucleus as regulating stem cell function, our study puts the focus on a different area," said Ding.

"E-cadherin is a protein on the cell surface that is very important to cell survival and cell growth."

The team found that when human embryonic stem cells are cut out from the colony, this key protein is disrupted and then internalized within the cell. Without e-cadherin on the cell surface, cell signalling between the cells and their environment is disrupted and the cells quickly die.

Both chemical compounds identified by the study, however, protected e-cadherin from damage.

In further experiments, the scientists found that the key difference between human and mouse embryonic stem cells lay not only within the cells themselves, but also in and controlled by their microenvironment — the surrounding cells, signalling factors, and extracellular matrix. The scientists were able to transfer human embryonic stem cells into a mouse embryonic stem cell microenvironment. There, the scientists found, human cells were more likely to survive, even without the survival-promoting compounds.

Moreover, when the scientists chemically induced human embryonic stem cells back to an earlier stage of development — which had an extracellular environment similar to mouse embryonic stem cells conventionally used in the laboratory — there were also no longer problems growing them in culture.

"This validated our mechanistic investigations from a different angle," said Ding, "showing that we had dissected out a very core regulatory mechanism."

Ding expects that the methods discussed in the new study will soon be widely adopted by stem cell laboratories around the world.

"My lab currently uses the novel small molecules indentified in this study on a routine basis, making our life significantly easier and advancing our efforts," said Ding.

"Even more, chemically inducing human embryonic stem cells back to an earlier stage of development has advantages for some areas of investigation."
.........

With few exceptions, all known forms of life on our planet rely on the same genetic code to specify the amino acid composition of proteins. Although different hypotheses abound, just how individual amino acids were assigned to specific three-letter combinations or codons during the evolution of the genetic code is still subject to speculation.

Taking their hints from relics of this evolution left behind in modern cells, researchers at the Salk Institute for Biological Studies concluded that after only two waves of "matching" and some last minute fiddling, all 20 commonly used amino acids were firmly linked with their respective codons, setting the stage for the emergence of proteins with unique, defined sequences and properties.

Analysis of ribosome structures, shown on the left, from four different species revealed a non-random affinity between anticodon-containing RNA triplets and their respective amino acids, shown on the right). Credit: Courtesy of David Johnson, Salk Institute for Biological Studies.

Their findings, which will be published in next week's online edition of the Proceedings of the National Academy of Sciences, provide the first in vivo data shedding light on the origin and evolution of the genetic code.

"Although different algorithms, or codes, were likely tested during a long period of chemical evolution, the modern code proved so robust that, once it was established, it gave birth to the entire tree of life," says the study's lead author Lei Wang, Ph.D., an assistant professor in the Chemical Biology and Proteomics Laboratory.

"But the universality of the code makes it very hard for researchers to study its formation since there are no organisms using a primitive or intermediate genetic code that we could analyze for comparison," he explains.

Cells provide a dazzling variety of functions that cover all of our body's needs, yet they make do with a very limited number of molecular building blocks. With few exceptions, all known forms of life use the same common 20 amino acids — and only those 20 — to keep alive organisms as diverse as humans, earthworms, tiny daisies, and giant sequoias.

Each of the 20 amino acids is matched to its own carrier molecule known as transfer RNA (tRNA). During protein synthesis, which is coordinated by so-called ribosomes, amino acids are brought out one by one by their respective tRNAs and inserted in the growing protein chain according to the instructions spelled out in the universal language of life — the genetic code. The code is "read" with the help of anticodons embedded in each tRNA, which pair up with their codon-counterparts.

Several hypotheses have been put forward to explain why codons are selectively assigned to specific amino acids.

"One of the theories, the stereochemical hypothesis, gained some traction when researchers could show that short codon- or anticodon-containing polynucleotide molecules like to interact with their respective amino acids," says graduate student and first author David B. F. Johnson.

If chemical or physical interactions between amino acids and nucleotide indeed drove the formation of the genetic code, Johnson reasoned, then he should be able to find relics of this mutual affinity in modern cells. He zoomed in on ribosomes, large complexes consisting of some 50 proteins interacting closely with ribosomal RNAs.

"Also, the ribosome emerged from an early evolutionary stage of life to help with the translation of the genetic code before the last universal common ancestor," explains Wang, "and therefore is more likely to serve as a molecular fossil that preserved biological evidence."

When Wang and Johnson probed bacterial ribosomes for imprints of the genetic code, they found evidence that direct interactions between amino acids and nucleotide triplet anticodons helped establish matching pairs.

"We now believe that the genetic code was established in two different stages," says Johnson.

Their data does not shed much light on the early code, consisting of prebiotically available amino acids — the kind generated in Stanley Miller's famous "zap"-experiment. But once some primitive translational mechanism had been established, new amino acids were added to the mix and started infiltrating the genetic code based on specific amino acid/anticodon interactions.

"We found evidence that a few amino acids were reassigned to a different codon but once the code was in place it took over," says Johnson.

"It might not have been the best possible solution but the only one that was viable at the time."
.........

Just one 'pulse' of artificial light at night disrupts the circadian mode of cell division Tuesday, 13 April 2010

Just one "pulse" of artificial light at night disrupts circadian cell division, reveals a new study carried out by Dr. Rachel Ben-Shlomo of the University of Haifa-Oranim Department of Environmental and Evolutionary Biology along with Prof. Charalambos P. Kyriacou of the University of Leicester.

"Damage to cell division is characteristic of cancer, and it is therefore important to understand the causes of this damage," notes Dr. Ben-Shlomo. The study has been published in the journal Cancer Genetics and Cytogenetics.

The current research was carried out by placing lab mice into an environment where they were exposed to light for 12 hours and dark for 12 hours. During the dark hours, one group of mice was given artificial light for one hour. Changes in the expression of genes in the rodents' brain cells were then examined.

Earlier studies that Dr. Ben-Shlomo carried out found that the cells' biological clock is affected, and in the present research, she revealed that the mode of cell division is also harmed and that the transcription of a large number of genes is affected. She states that it is important to note that those genes showing changes in their expression included genes that are connected to the formation of cancer as well as genes that assist in the fight against cancer.

"What is certain is that the natural division is affected," Dr. Ben-Shlomo clarifies.

This research joins earlier studies from the University of Haifa on the effects of exposure to artificial light at night.
.........

An innovative treatment using stem cells undergoes testing at the research centre of the CHUM, in collaboration with the Maisonneuve-Rosemont HospitalTuesday, 13 April 2010

Some patients with heart muscles seriously affected by coronary heart disease may soon be able to benefit from an innovative treatment. Researchers at the Research Centre of the Centre hospitalier de l'Université de Montréal (CRCHUM), in collaboration with the Maisonneuve-Rosemont Hospital (MRH) are evaluating the safety, feasibility and efficacy of injecting stem cells into the hearts of patients while they are undergoing coronary bypass surgery. These stem cells could improve healing of the heart and its function.

The IMPACT-CABG (implantation of autologous CD133+ stem cells in patients undergoing coronary artery bypass grafting) protocol evaluates this experimental procedure, which is destined for patients suffering from ischemic heart disease, in which the blood supply to the heart is decreased and associated with heart failure. These patients undergo open-heart coronary bypass surgery, performed by the medical team to improve perfusion of the heart muscle. A few weeks ago, the first patient received progenitor CD133+ stem cells isolated from his bone marrow and enriched at the Cell Therapy Laboratory of the MRH, and has been doing very well ever since. Already, improvement has been noted in the contraction capacity of his heart, which has improved its ability to pump blood.

Objective of the intervention
The IMPACT-CABG study targets a group of patients who suffer heart muscle failure due to coronary heart disease. The goal is to add another treatment option to coronary bypass to promote healing and regeneration of the damaged heart muscle. This new procedure is less invasive and less expensive than heart transplant, the only treatment now available for patients with severe heart failure. The researchers plan to recruit a total of 20 patients throughout Québec in the first phase. A second Canadian centre, at the General Hospital of the University of Toronto, will also take part in the trial. In 2007, the CRCHUM, in collaboration with the MRH, began the COMPARE-AMI clinical trial, to evaluate the safety and feasibility of intramyocardial injection of stem cells (injecting them into the heart through a catheter) in a different group of patients who have suffered their first infarction.

A Canadian first
Before the IMPACT-CABG trial, previous studies in other countries had also evaluated the safety and feasibility of injecting different stem cells in the hearts of patients with cardiac dysfunction. This is a first study in Canada evaluating intramyocardial injection of stem cells.

"Also, no research team in the country had implemented such a complete treatment process, going from harvesting stem cells in the patient, treating them, and injecting them directly into the myocardium," states Dr. Nicolas Noiseux, cardiac surgeon at the CHUM and principal investigator in the study.

"Moreover, the methods used to evaluate the recovery of heart function make use of cutting-edge imaging techniques," reports Dr. Samer Mansour, cardiologist at the CHUM, principal co-investigator.

To prepare for the intervention, cells from the bone marrow harvested at the CHUM are transferred to the cell therapy laboratory of the MRH to isolate the most immature stem cells, which will be injected directly into the patient's heart.

"MRH has developed a unique expertise in cellular therapy. This expertise serves cardiology in those specific cases and we hope to develop other applications to regenerate the cornea, cartilage and other tissues and organs," adds Dr. Denis Claude Roy, haematologist, Director of research and Director, Cell therapy Laboratory at MRH, and also a principal co-investigator.
.........

Friday, 9 April 2010

Researchers at the Cedars-Sinai Heart Institute have found in animals that infusing cardiac-derived stem cells with micro-size particles of iron and then using a magnet to guide those stem cells to the area of the heart damaged in a heart attack boosts the heart's retention of those cells and could increase the therapeutic benefit of stem cell therapy for heart disease.

Circulation Research, a scientific journal of the American Heart Association, publishes the study today online. The study also will appear in the journal's May 28th printed edition.

"Stem cell therapies show great promise as a treatment for heart injuries, but 24 hours after infusion, we found that less than 10 percent of the stem cells remain in the injured area," said Eduardo Marbán, M.D., director of the Cedars-Sinai Heart Institute.

"Once injected into a patient's artery, many stem cells are lost due to the combination of tissue blood flow, which can wash out stem cells, and cardiac contraction, which can squeeze out stem cells. We needed to find a way to guide more of the cells directly to the area of the heart that we want to heal."

Marbán's team, including Ke Cheng, Ph.D. and other researchers, then began a new animal investigation, loading cardiac stem cells with micro-size iron particles. The iron-loaded cells were then injected into rats with a heart attack. When a toy magnet was placed externally above the heart and close to the damaged heart muscle, the stem cells clustered at the site of injury, retention of cells in the heart tripled, and the injected cells went on to heal the heart more effectively.

"Tissue viability is enhanced and heart function is greater with magnetic targeting," said Marbán, who holds the Mark Siegel Family Foundation Chair at the Cedars-Sinai Heart Institute and directs Cedars-Sinai's Board of Governors Heart Stem Cell Center.

"This remarkably simple method could easily be coupled with current stem cell treatments to enhance their effectiveness."

In the future, this finding in the animal model may build on the ongoing, groundbreaking clinical trial led by Raj Makkar, M.D., director of interventional cardiology for the Cedars-Sinai Heart Institute. In the clinical trial, which is based on Marbán's research, heart attack patients undergo two minimally-invasive procedures in an effort to repair and re-grow healthy muscle in a heart injured by a heart attack. First, a biopsy of each patient's own heart tissue is used to grow specialized heart stem cells. About a month later, the multiplied stem cells are then injected back into the patient's heart via a coronary artery.

The two-step procedure was completed on the first patient in June 2009. Complete results are expected in early-2011.

Recently, Marbán received a $5.5 million grant from the California Institute for Regenerative Medicine to continue developing cardiac stem cell therapies.

The Cedars-Sinai Heart Institute is internationally recognized for outstanding heart care built on decades of innovation and leading-edge research. From cardiac imaging and advanced diagnostics to surgical repair of complex heart problems to the training of the heart specialists of tomorrow and research that is deepening medical knowledge and practice, the Cedars-Sinai Heart Institute is known around the world for excellence and innovations.

Marbán invented the methods used to grow and expand stem cells from heart biopsies. Marbán filed patents regarding those innovations which are licensed by Capricor, Inc. Marbán and his wife, Linda Marban, Ph.D. are both founders of Capricor, Inc. Dr. Eduardo Marban serves on its Board of Directors, and owns equity in the company. Dr. Linda Marban serves as a consultant to Capricor.
.........